A3 adenosine receptor ligands for modulation of pigmentation

ABSTRACT

The present invention relates to compositions and methods for modulating melanin production, secretion and/or accumulation in human skin cells. In particular, the present invention relates to the use of A3 adenosine receptor antagonists in compositions and methods for the treatment and amelioration of hyper-pigmentation conditions and for the lightening of skin, and to the use of A3 adenosine receptor agonists in compositions and methods for the treatment and amelioration of hypo-pigmentation conditions and for the tanning of skin.

FIELD OF THE INVENTION

The present invention relates to pharmaceutical and cosmeticcompositions comprising A3 adenosine receptor ligands and methods of usethereof for modulating melanin production, secretion and/or accumulationin skin.

BACKGROUND OF THE INVENTION

A variety of dermatological compositions have been suggested for skinwhitening to counteract abnormal pigmentation occurring in variousdisorders. Such disorders include post inflammatory hyperpigmentation,(attributed to various preceding conditions such as infections, allergicreactions, mechanical injuries, reactions to medications, phototoxiceruptions, trauma for example burns, and inflammatory diseases such aslichen planus, lupus erythematosus, atopic dermatitis), melasma,periorbital darkening, pigmented keratosis, lentigo senilis, ephelides,chloasma, café au lait spots, liver spots, freckles and lesions observedin Addison's disease, hemochomatosis, piebaldism, and the like. Inaddition, there is an enormous demand within populations withgenetically inherited dark skin to lighten their skin color.

In mammals, skin and hair color is primarily determined by the amount ofmelanin pigments that are synthesized by melanocytes within specializedorganelles called melanosomes. There is no evidence for differences inmelanosome biogenesis between follicular and epidermal melanocytes.Thus, in black hair follicles, melanocytes contain the largest numberand most electron-dense melanosomes. In brown hair, bulb melanocytes aresmaller, and in blonde hair melanosomes are poorly melanized (Slominskiet al., 2004). Melanin synthesis (also termed melanogenesis) iscontrolled by at least three enzymes: tyrosinase,tyrosinase-related-protein 1 (TRP1) and tyrosinase-related-protein 2(TRP2), among which tyrosinase has the key role of catalyzing the ratelimiting steps of the hydroxylation reaction of tyrosine to L-DOPA andthe oxidation reaction of L-DOPA into dopaquinone.

Melanogenesis and melanosome transfer from the melanocytes to theneighboring keratinocytes involve a complex network of regulatoryprocesses, and are induced primarily by ultraviolet radiation. Solarradiation acts either directly on melanocytes or indirectly though therelease of keratinocyte-derived melanocyte stimulating hormone (α-MSH),a potent inducer of melanogenesis both in vivo and in vitro (Englaro etal., 1998). α-MSH stimulates adenylate cyclase activity leading to anincrease in cAMP level which elevates the expression of melanogenicenzymes, in particular that of tyrosinase (Hunt of al., 1994; Im et al.,1998; Lee et al., 2007). In contrast, inhibition of adenylate cyclaseactivity resulting in decreased levels of intracellular cAMP isassociated with inhibition of melanogenesis and depigmentation (Choi etal., 2008; Lee et al., 2007). Keratinocyte growth factor (KGF), aparacrine mediator of human keratinocyte growth and differentiation, hasbeen shown to induce melanosome transfer from melanocytes tokeratinocytes through a phagocytic process (Cardinali et al., 2005).

Dermatological compositions which have been disclosed for hair and skinbleaching typically act by destroying or disrupting melanin granules,inhibiting melanin formation (such as by inhibiting tyrosinase ormelanocyte activity), or both. Various skin whitening compositions aredisclosed for example, in U.S. Pat. Nos. 5,980,904, 5,747,006, and6,077,503. Many of these compositions contain harsh chemicals such asperoxides, acids or formaldehyde, or thiolated compounds such asglutathione, cysteine, mercaptosuccinic acid, mercaptodextran, andmercaptoethanol, which have an objectionable odor that makes productscontaining them undesirable for use

Hydroquinone for topical application is approved in the United Statesfor non-prescription use, and acts by suppressing melanocyte activity.U.S. Pat. No. 6,068,834 discloses hydroquinone-based compounds andcompositions Hydroquinone, however, is oxidized by air, light, andtyrosinase itself, which adversely affects the shelf life ofpreparations containing it and its bioavailability upon application.Hydroquinone can cause burning, redness, sensitization and irritation insome individuals, particularly after application of quantitiessufficient to cause skin bleaching. Oxidized products of hydroquinonehave also been implicated in skin irritation and pigmentation rebound.

Retinoids and corticosteroids for topical use have been suggested ashypopigmenting agents, as have laser treatment and chemical peels, butthese fall short of desirable responses. A combination therapycontaining tretinoin and fluocinolone with hydroquinone has beendisclosed (Willis, 2000). Kojic acid and arbutin have also beensuggested (Draelos, 2007), but these weak tyrosinase inhibitorsgenerally display low bioavailability. Furthermore, arbutin isineffective at levels allowed by Japanese quasi-drug regulations, andkojic acid has been banned for quasi-drug usage in Japan due to itsmutagenic properties (Boissy et al., 2005).

U.S. Pat. No. 7,019,029 discloses the use of hydroxythtronic acidderivatives alone or in combination with tetronic acid, hydroquinone,glycolic acid and/or ascorbyl palmitate as skin whiteners.

Various plant extracts have been disclosed for bleaching skin or forenhancing the appearance of fair skin, which have in some cases beenused for centuries in Asia or Europe. U.S. Pat. No. 5,602,259 disclosesa furanone extracted from Pinaceae plants for use in whitening skin byinhibiting melanin formation.

U.S. Pat. No. 5,905,091 discloses a composition comprising a carrier anda prostaglandin, and optionally comprising a lysosomotropic agent, aphosphodiesterase inhibitor, and/or methylxanthines, and a method of usethereof for stimulating synthesis of melanin in a human melanocytethereby enhancing pigmentation of human skin,.

U.S. Pat. No. 5,554,359 discloses a composition comprising alysosomotropic agent, and optionally phosphodiesterase inhibitors,and/or methylxanthines for increasing synthesis of melanin in a humanmelanocyte thereby enhancing pigmentation of human skin.

Adenosine is an endogenous purine nucleoside ubiquitous in mammaliancells, which is an important regulatory compound that mediates manyphysiological effects via binding to its specific A1, A2 and A3 cellsurface receptors. Interaction of adenosine with its receptors initiatessignal transduction pathways, in particular that of the adenylatecyclase effector system, which utilizes cAMP as a second messenger. TheA1 and A3 adenosine receptors, which are coupled to Gi proteins, inhibitadenylate cyclase and lead to a decrease in the level of intracellularcAMP, while the A2 adenosine receptor, which is coupled to Gs proteins,activates adenylate cyclase, thereby increasing cAMP levels (see forexample, Fredholm et al., 2000).

The use of various adenosine receptor agonists and antagonists fortreatment of different disease states and pathologies has beensuggested, including for example, inflammation (Salvatore et al., 2000),neurodegeneration (Von Lubitz, 1999), asthma (Forsythe and Ennis, 1999),cardiac ischemia (Liang and Jacobson, 1998), and tumors (Yao et al.,1997).

U.S. Pat. No. 7,064,112 discloses the use of A3 adenosine receptoragonists for the prevention and treatment of leukopenia, and for theinhibition of abnormal cell growth and proliferation of tumor cells.

WO 2008/023362 discloses methods and compositions useful for treatingcancer based on the combination of methotrexate and an A3 adenosinereceptor agonist.

U.S. Pat. No. 7,465,715 discloses the use of A3 adenosine receptoragonists for the treatment of multiple sclerosis.

U.S. Pat. No. 7,465,715 discloses the use of A3 adenosine receptoragonists for inhibiting viral replication in cells.

U.S. Pat. No. 7,141,553 discloses the use of A3 adenosine receptoragonists for the treatment of inflammatory arthritis.

U.S. Pat. No. 5,998,423 discloses the use of adenosine A1 receptorantagonists and of adenosine A2 receptor agonists for increasing melaninproduction in skin or hair, and the use of adenosine A1 receptoragonists and adenosine A2 receptor antagonists for decreasing melaninproduction in skin or hair.

U.S. Patent Application Publication No. 2002/0115635 discloses atreatment method comprising administering to a subject an active agentselected from the group consisting of an adenosine A1 receptor ligand,an A2 adenosine receptor ligand, an adenosine A3 receptor ligand and acombination thereof. The method is disclosed to be effective for avariety of diseases and disorders, inter alia hair loss, which requireelevation of GSK-313 activity for treatment.

U.S. Patent Application Publication No. 2007/0299032 discloses a methodfor maintaining and promoting hair thickening comprising applying to thescalp an external skin preparation containing one or more types ofagents, inter alia the A3 adenosine receptor agonist Cl-IB-MECA.

U.S. Patent Application Publication No. 2008/0044439 discloses use ofp38 inhibitors for treating skin conditions such as vitiligo, anddescribes adenosine A3 antagonists as possible p38 inhibitors.

There remains an unmet need for new compositions which modulate melaninproduction and thereby lighten the skin or hair or promote pigmentationfor both pharmaceutical and cosmetic purposes.

SUMMARY OF THE INVENTION

The present invention provides methods as well as pharmaceutical andcosmetic compositions useful for modulating the production, secretionand/or accumulation of melanin in skin, in particular within skin cellssuch as melanocytes and keratinocytes, thereby modulating pigmentationof the skin and hair. In particular, the present invention provides useof A3 adenosine receptor antagonists in compositions and methods for thetreatment and amelioration of hyper-pigmentation skin conditions and forcosmetic lightening of skin and hair color, as well as use of A3adenosine receptor agonists in compositions and methods for thetreatment and amelioration of hypo-pigmentation skin conditions and forcosmetic skin tanning.

The present invention is based in part on the surprising discovery thatactivation of the A3 adenosine receptor causes an increase inpigmentation, while deactivation of the A3 adenosine receptor causes adecrease in pigmentation. The present invention is highly unexpectedover the prior art, which teaches that activation of the A3 adenosinereceptor, which is known to be negatively coupled to adenylate cyclase,should inhibit melanogenesis due to decreased intracellular levels ofcAMP and thus lead to depigmentation.

Surprisingly and in contrast to the teachings of the prior art, theinventors of the present invention have shown that melanocytes treatedwith the A3 adenosine receptor agonist IB-MECA unexpectedly exhibit anincrease in pigmentation, attributable to an increase in melaninsecretion by the melanocytes. Similarly, the inventors of the presentinvention have shown that treatment of melanoctyes with the A3 adenosinereceptor antagonist MRS-1523, unexpectedly led to depigmentation.

Moreover, the inventors of the present invention have found that A3adenosine receptor ligands may exert modulation of pigmentation byaltering melanin production and/or secretion in melanocytes, as well asaltering melanin accumulation in keratinocytes and/or melanin secretionfrom melanocytes to keratinocytes.

Without wishing to be bound by any theory or mechanism of action, it iscontemplated that the effect of A3 adenosine receptor agonists inenhancing pigmentation and the effect of A3 adenosine receptorantagonists in decreasing pigmentation, may respectively involve Mitfactivation and Mitf downregulation, and mediation thereof via the Aktand ERK signaling pathways.

According to a first aspect, the present invention provides a method formodulating melanin production, secretion, accumulation or a combinationthereof in at least one skin cell, comprising the step of contacting theskin cell with a compound selected from the group consisting of an A3adenosine receptor antagonist and an A3 adenosine receptor agonist in anamount effective to modulate melanin production, secretion, and/oraccumulation in the skin cell.

In particular embodiments, the at least one skin cell is a melanocyte, akeratinocyte, or tissue comprising a plurality of melanocytes and/orkeratinocytes.

In a particular embodiment, the compound is an A3 adenosine receptoragonist, and the modulating comprises increasing at least one of melaninproduction, secretion or accumulation. Each possibility represents aseparate embodiment of the invention.

In a particular embodiment, the skin cell is a melanocyte, and the A3adenosine receptor agonist is in an amount effective to increase atleast one of melanin production and melanin secretion in saidmelanocyte. Each possibility represents a separate embodiment of theinvention.

In a particular embodiment, the skin cell is a keratinocyte, and the A3adenosine receptor agonist is in an amount effective to increaseaccumulation of melanin in said keratinocyte. In a particularembodiment, the A3 adenosine receptor agonist is in an amount effectiveto increase secretion of melanin from a melanocyte to a keratinocyte.

According to particular embodiments, the A3 adenosine receptor agonisthas a binding affinity (Ki) for the A3 adenosine receptor that is lessthan about 200 nM, preferably less than about 100 nM, more preferablyless than about 50 nM, even more preferably less than about 10 nM. Eachpossibility represents a separate embodiment of the invention. Accordingto particular embodiments, the binding affinity of the A3 adenosinereceptor agonist for the adenosine A3 receptor is at least 20 timesgreater than the binding affinity of said agonist for the A1 adenosinereceptor.

According to particular embodiments, the A3 adenosine receptor is thehuman A3 adenosine receptor.

According to particular embodiments, the A3 adenosine receptor agonistis a selective A3 adenosine receptor agonist.

According to particular embodiments of the invention disclosed herein,the A3 adenosine receptor agonist is anN⁶-substituted-adenosine-5′-uronamide. In some embodiments, theN⁶-substituted-adenosine-5′-uronamide is selected from the groupconsisting of an N⁶-monosubstituted-adenosine-5′-uronamide and anN⁶-disubstituted-adenosine-5′-uronamide. According to particularembodiments, the A3 adenosine receptor agonist is selected from thegroup consisting of an N⁶-benzyladenosine-5′-uronamide; anN⁶-4-substituted-sulfonamidophenylcarbamoyl-adenosine-5′-uronamide; a2-chloro-N⁶-substituted-4′-thioadenosine-5′-uronamide; an(N)-methanocarba adenosine-5′-uronamide and derivatives and analogsthereof. Each possibility represents a separate embodiment of thepresent invention.

According to particular embodiments, the A3 adenosine receptor agonistis selected from the group consisting of N⁶-(3-iodobenzyl)adenosine-5′-(N-methyluronamide) (IB-MECA);2-chloro-N⁶-(3-iodobenzyl)-adenosine-5′-(N-methyluronamide)(Cl-IB-MECA); N⁶-(4-amino-3-iodobenzyl)-adenosine-5′-(N-methyluronamide)(AB-MECA); N⁶-2-(4-aminophenyl) ethyladenosine (APNEA);2-(1-hexynyl)-N-methyladenosine,2-chloro-N⁶-(3-iodobenzyl)-9-[5-(methylcarbamoyl)-D-ribofuranosyl]adenine(CF-102);(2S,3S,4R,5R)-3-amino-5-[6-(2,5-dichlorobenzylamino)purin-9-yl]-4-hydroxytetrahydrofuran-2-carboxylicacid methylamide (CP-532,903);2-chloro-4′-thioadenosine-5′-methyluronamide;(1′R,2′R,3′S,4′R,5′S)-4-{2-chloro-6-[(3-chlorophenylmethyl)amino]purin-9-yl}-1-(methylaminocarbonyl)bicyclo[3.1.0]hexane-2,3-diol (MRS-3558), and derivatives and analogs thereof. Eachpossibility represents a separate embodiment of the present invention.

In a particular embodiment, the A3 adenosine receptor agonist isselected from the group consisting of N⁶-(3-iodobenzyl)adenosine-5′-(N-methyluronamide) (IB-MECA);2-chloro-N⁶-(3-iodobenzyl)-adenosine-5′-(N-methyluronamide)(Cl-IB-MECA); N⁶-(4-amino-3-iodobenzyl)-adenosine-5′-(N-methyluronamide)(AB-MECA); and N⁶-2-(4-aminophenyl) ethyladeno sine (APNEA).

In a particular embodiment, the A3 adenosine receptor agonist isselected from the group consisting of N⁶-(3-iodobenzyl)adenosine-5′-(N-methyluronamide) (IB-MECA);2-chloro-N⁶-(3-iodobenzyl)-adenosine-5′-(N-methyluronamide)(Cl-IB-MECA); 2-(1-hexynylhexynyl)-N-methyladenosine;2-chloro-N⁶-(3-iodobenzyl)-9-[5-(methylcarbamoyl)-D-ribofuranosyl]adenine(CF-102); and(2S,3S,4R,5R)-3-amino-5-[6-(2,5-dichlorobenzylamino)purin-9-yl]-4-hydroxytetrahydrofuran-2-carboxylicacid methyl amide (CP-532,903).

In a particular embodiment, the A3 adenosine receptor agonist isselected from the group consisting of N⁶-(3-iodobenzyl)adenosine-5′-(N-methyluronamide) (IB-MECA) and2-chloro-N⁶-(3-iodobenzyl)-adenosine-5′-(N-methyluronamide)(Cl-IB-MECA).

According to a currently preferred embodiment, the A3 adenosine receptoragonist is N⁶-(3-iodobenzyl) adenosine-5′-(N-methyluronamide) (IB-MECA).

According to another embodiment, the method for increasing at least oneof melanin production, secretion or accumulation further comprisescontacting the skin cell with an allosteric modulator of the A3adenosine receptor. In particular embodiments, the allosteric modulatoris an allosteric enhancer of the A3 adenosine receptor.

In a particular embodiment, the skin cell is in a human subject having ahypo-pigmentation disorder selected from the group consisting ofvitiligo, piebaldism, leukoderma due to cicatrisation, nevusdepigmentosis and depigmentation due to a skin graft procedure.

According to another particular embodiment of the method disclosedherein, the compound is an A3 adenosine receptor antagonist, and themodulating comprises decreasing at least one of melanin production,secretion or accumulation in the skin cell. Each possibility representsa separate embodiment of the invention.

According to a particular embodiment, the skin cell is a melanocyte, andthe A3 adenosine receptor antagonist is in an amount effective todecrease at least one of melanin production and melanin secretion insaid melanocyte. Each possibility represents a separate embodiment ofthe invention.

According to a particular embodiment, the skin cell is a keratinocyte,and the A3 adenosine receptor antagonist is in an amount effective todecrease accumulation of melanin in said keratinocyte. According to aparticular embodiment, the A3 adenosine receptor antagonist is in anamount effective to decrease secretion of melanin from a melanocyte to akeratinocyte.

According to particular embodiments, the A3 adenosine receptorantagonist has a binding affinity (Ki) for the A3 adenosine receptorthat is less than about 200 nM, preferably less than about 100 nM, morepreferably less than about 50 nM, even more preferably less than 10 nM.Each possibility represents a separate embodiment of the, invention.

According to particular embodiments, the binding affinity of the A3adenosine receptor antagonist for the adenosine A3 receptor is at least20 times greater than the binding affinity of said antagonist for the A1adenosine receptor.

According to particular embodiments, the A3 adenosine receptor is thehuman A3 adenosine receptor. According to some embodiments, the A3adenosine receptor antagonist is a selective A3 adenosine receptorantagonist.

According to a particular embodiment, the A3 adenosine receptorantagonist is a compound selected from the group consisting of adihydropyridine, a pyridine, a pyridinium salt, a triazoloquinazoline,an imidazoquinoline, a triazolopurine, an N⁶-substituted-7-deazapurine,and derivatives and analogs thereof. Each possibility represents aseparate embodiment of the present invention.

According to particular embodiments, the A3 adenosine receptorantagonist is selected from the group consisting of3-propyl-6-ethyl-5[(ethylthio)carbonyl]-2-phenyl-4-propyl-3-pyridinecarboxylate (MRS-1523);1,4-dihydro-2-methyl-6-phenyl-4-(phenylethynyl)-3,5-pyridinedicarboxylicacid 3-ethyl-5-[(3-nitrophenyl)methyl]ester (MRS-1334);3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(±)-dihydropyridine-3,5dicarboxylate (MRS-1191); 3-ethyl5-benzyl-2-methyl-6-phenyl-4-styryl-1,4-(±)-dihydropyridine-3,5-dicarboxylate(MRS-1097);5-n-butyl-8-(4-trifluoromethylphenyl)-3H-[1,2,4]triazolo-[5,1-i]purineOT-7999;(2R,3R,4S,5S)-2-[N⁶-3-iodobenzyl)adenos-9′-yl]-7-aza-1-oxa-6-oxospiro[4,4]-nonan-4,5-diol(MRS-1292); N-(2-methoxyphenyl)-N′-[2-(3-pyridinyl)-4-quinazolinyl]-urea(VUF-5574);(8R)-8-ethyl-1,4,7,8-tetrahydro-4-5H-imidazo[2,1-i]purin-5-one (PSB-11);2-phenoxy-6-(cyclohexylamino)purine (MRS-3777);5N-(4-methoxyphenylcarbamoyl)amino-8-propyl-2-(2-furyl)pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (MRE3008F20) and derivatives andanalogs thereof. Each possibility represents a separate embodiment ofthe present invention.

In a particular embodiment, the A3 adenosine receptor antagonist isselected from the group consisting of3-propyl-6-ethyl-5[(ethylthio)carbonyl]-2-phenyl-4-propyl-3-pyridinecarboxylate (MRS-1523);1,4-dihydro-2-methyl-6-phenyl-4-(phenylethynyl)-3,5-pyridinedicarboxylicacid 3-ethyl-5-[(3-nitrophenyemethyl] ester (MRS-1334); and3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(±)-dihydropyridine-3,5dicarboxylate (MRS-1191).

According to a currently preferred embodiment, the A3 adenosine receptorantagonist is3-propyl-6-ethyl-5[(ethylthio)carbonyl]-2-phenyl-4-propyl-3-pyridinecarboxylate (MRS-1523).

According to particular embodiments of the method for decreasing atleast one of melanin production; secretion or accumulation, the methodfurther comprises the step of contacting the skin cell with at least oneof hydroxytetronic acid; tetronic acid; hydroquinone; an α-hydroxy acid,a fatty acid ester of ascorbic acid; a tyrosinase inhibitor and atyrosine phosphatase inhibitor.

In a particular embodiment, the skin cell is in a human subject having ahyper-pigmentation disorder selected from the group consisting ofpigmented spots, lentigo senilis, freckles, café au lait spots, liverspots, ephelides, periorbital darkening, post-inflammatoryhyper-pigmentation, pigmented keratosis, melasma, chloasma, andhyper-pigmentation due to a skin graft procedure.

In another aspect, the invention further provides a compound selectedfrom the group consisting of an A3 adenosine receptor antagonist and anA3 adenosine receptor agonist for use in modulating melanin production,secretion, accumulation or a combination thereof in at least one skincell.

In a particular embodiment, the compound is an A3 adenosine receptoragonist in an amount effective for increasing at least one of melaninproduction, secretion or accumulation. In another particular embodiment,the compound is an A3 adenosine receptor antagonist in an amounteffective for decreasing at least one of melanin production, secretionor accumulation. Each possibility represents a separate embodiment ofthe invention.

According to another aspect, the present invention provides a cosmeticor pharmaceutical composition for use in modulating at least one ofmelanin production, secretion or accumulation in skin, wherein thecomposition comprises as an active ingredient an A3 adenosine receptorantagonist or an A3 adenosine receptor agonist; and a pharmaceuticallyacceptable carrier. In a particular embodiment the pharmaceuticallyacceptable carrier is capable of delivering the active ingredient to askin cell under in vivo conditions.

According to one embodiment, the composition comprises an A3 adenosinereceptor agonist as described herein, in an amount effective forincreasing at least one of melanin production, secretion or accumulationin skin. Each possibility represents a separate embodiment of theinvention.

According to a particular embodiment, the composition comprises an A3adenosine receptor agonist in an amount effective for increasing atleast one of melanin production and melanin secretion in a melanocyte.Each possibility represents a separate embodiment of the invention.

According to a particular embodiment, the composition comprises an A3adenosine receptor agonist in an amount effective for increasing melaninaccumulation in a keratinocyte. According to a particular embodiment,the composition comprises an A3 adenosine receptor agonist in an amounteffective for increasing melanin secretion from a melanocyte to akeratinocyte.

According to a particular embodiment, the composition comprisesN⁶-(3-iodobenzyl)adenosine-5′-(N-methyluronamide) (IB-MECA) as the A3adenosine receptor agonist.

According to another embodiment, the composition comprising an A3adenosine receptor agonist, further comprises an allosteric enhancer ofthe A3 adenosine receptor.

According to another embodiment, the composition comprises an A3adenosine receptor antagonist as described herein, in an amounteffective for decreasing at least one of melanin production, secretionor accumulation in skin. Each possibility represents a separateembodiment of the invention.

According to a particular embodiment, the composition comprises an A3adenosine receptor antagonist in an amount effective for decreasing atleast one of melanin production and melanin secretion in a melanocyte.Each possibility represents a separate embodiment of the invention.

According to a particular embodiment, the composition comprises an A3adenosine receptor antagonist in an amount effective for decreasingmelanin accumulation in a keratinocyte. According to a particularembodiment, the composition comprises an A3 adenosine receptorantagonist in an amount effective for decreasing melanin secretion froma melanocyte to a keratinocyte.

According to a particular embodiment, the composition comprises3-propyl-6-ethyl-5[(ethylthio)carbonyl]-2-phenyl-4-propyl-3-pyridinecarboxylate (MRS-1523) as the A3 adenosine receptor antagonist.

According to another embodiment, the composition comprising an A3adenosine receptor antagonist further comprises at least one ofhydroxytetronic acid; tetronic acid; hydroquinone; an α-hydroxy acid, afatty acid ester of ascorbic acid; a tyrosinase inhibitor and a tyrosinephosphatase inhibitor.

According to particular embodiments, the compositions are formulated foradministration by a route selected from the group consisting of directadministration into, onto or in the vicinity of a skin site ofhyper-pigmented or hypo-pigmented tissue, topical, intradermal,transdermal, subcutaneous, intramuscular, intraperitoneal, intravenous,subcutaneous, intranasal and oral administration.

According to another aspect, the present invention provides a method fortreating or ameliorating a hyper-pigmentation condition of human skin,the method comprising administering to a human subject in need thereofan effective amount of a cosmetic or pharmaceutical compositioncomprising as an active ingredient an A3 adenosine receptor antagonistaccording to the principles of the invention.

According to particular embodiments, the hyper-pigmentation condition isselected from the group consisting of pigmented spots, lentigo senilis,freckles, café au lait spots, liver spots, ephelides, periorbitaldarkening, post-inflammatory hyper-pigmentation, pigmented keratosis,melasma, chloasma and hyper-pigmentation due to a skin graft procedure.In a particular embodiment the composition is a cosmetic composition forlightening dark skin.

According to another aspect, there is provided a cosmetic orpharmaceutical composition comprising an A3 adenosine receptorantagonist for use in treating or ameliorating a hyper-pigmentationcondition of human skin, in accordance with the invention.

According to yet another aspect, the present invention provides a methodfor treating or ameliorating a hypo-pigmentation condition of human skincomprising administrating to a human subject in need thereof aneffective amount of a cosmetic or pharmaceutical composition comprisingas an active ingredient an A3 adenosine receptor agonist according tothe principles of the invention. According to particular embodiments,the hypo-pigmentation condition is selected from the group consisting ofpigmented vitiligo, piebaldism, leukoderma due to cicatrisation, nevusdepigmentosis, and depigmentation due to a skin graft procedure. In aparticular embodiment the composition is a cosmetic composition fortanning fair skin.

In particular embodiments, the administering comprises topicallyapplying the composition comprising the A3 adenosine receptor agonist orthe A3 adenosine receptor antagonist to skin of the subject. Inparticular embodiments, the A3 adenosine receptor agonist or the A3adenosine receptor antagonist is topically administered over a period ofat least two weeks, or at least one month, or at least two months.

According to another aspect, there is provided a cosmetic orpharmaceutical composition comprising an A3 adenosine receptor agonistfor use in treating or ameliorating a hypo-pigmentation condition ofhuman skin, in accordance with the invention.

According to additional embodiments, the administering of the cosmeticor pharmaceutical composition to the subject is carried out by a routeselected from the group consisting of direct administration into, ontoor in the vicinity of hyper-pigmented or hypo-pigmented skin, topical,intradermal, transdermal, subcutaneous, intramuscular, intraperitoneal,intravenous, subcutaneous, intranasal and oral administration. Accordingto certain preferred embodiments, the composition is topicallyadministered.

According to further embodiments, the cosmetic or pharmaceuticalcomposition is in a form selected from the group consisting of asolution, a suspension, an emulsion, a cream, a gel, an aerosolformulation and a sustained-release formulation.

Further embodiments and the full scope of applicability of the presentinvention will become apparent from the detailed description givenhereinafter. However, it should be understood that the detaileddescription and specific examples, while indicating preferredembodiments of the invention, are given by way of illustration only,since various changes and modifications within the spirit and scope ofthe invention will become apparent to those skilled in the art from thisdetailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the effect of the A3 adenosine receptor agonist IB-MECA onmelanin synthesis and secretion in B16 melanocytes. Cells were culturedin 10% DMEM in the presence of various concentrations of IB-MECA for 5days. Levels of melanin in the media and in the cell lysates, and totalprotein content in the cell lysates were then determined as described inMaterials and Methods. Melanin levels were measured and calculatedseparately for each sample.

FIG. 1A shows melanin levels in cell lysates and in the media expressedas μg melanin/mg protein.

FIG. 1B shows total melanin levels expressed as percent of control. Eachvalue is expressed as the mean±SD, n=3 *p<0.05, **p<0.001 compared tothe control.

FIG. 1C is a photograph showing the dose response of melanin secretionto the media by cells treated with the indicated concentrations ofIB-MECA.

FIG. 2 shows the effect of the A3 adenosine receptor antagonist MRS-1523(abbreviated as “MRS”) on melanin synthesis and secretion in B16melanocytes. Cells were cultured in 10% DMEM in the presence of variousconcentrations of MRS-1523 for 5 days. Levels of melanin in the mediaand in the cell lysates, and total protein content in the cell lysateswere then determined as described in Materials and Methods. Melaninlevels were measured and calculated separately for each sample.

FIG. 2A shows melanin levels in cell lysates and in the media expressedas μg melanin/mg protein.

FIG. 2B shows total melanin levels expressed as percent of control. Eachvalue is expressed as the mean+SD, n=3 *p<0.05, **p<0.001 compared tothe control.

FIG. 3 shows the time course of melanin synthesis and secretion by B16melanocytes treated with IB-MECA or MRS-1523. Cells were exposed to 10μM of IB-MECA or MRS-1523 and the melanin was monitored at differenttime intervals. Levels of melanin in the media and in the cell lysates,and total protein content in the cell lysates were determined asdescribed in Materials and Methods. Melanin levels were measured andcalculated separately for each sample.

FIG. 3A is a photograph of the cells following treatment with theindicated compounds for 24 h. Arrows indicate positions of melanosomes.N, nucleus.

FIG. 3B depicts transfer of melanosomes from one melanocyte to the otherby a dendrite-dendrite interaction. Arrow indicates position ofmelanosomes.

FIG. 3C shows total melanin levels after various treatment intervalsexpressed as percent of control.

FIG. 3D shows melanin levels expressed as μg melanin/mg protein in celllysates and in the media following exposure of melanocytes to IB-MECA orMRS-1523 for 5-days. For FIGS. 3C and 3D, each value is expressed as themean±SD, n=3 *p<0.05, **p<0.001 compared to the control.

FIG. 3E is a photograph showing melanin secretion to the media following5 days of exposure to IB-MECA or MRS-1523, as indicated.

FIG. 4 shows the effect of kojic acid on melanin synthesis andsecretion. B16 melanocytes were cultured in 10% DMEM in the presence ofKojic acid for 5 days. Levels of melanin in the media and in the celllysates, and total protein content in the cell lysates were determinedas described in Materials and Methods. Melanin levels were measured andcalculated separately for each sample.

FIG. 4A shows melanin levels in cell lysates and in the media expressedas μg melanin/mg protein.

FIG. 4B shows total melanin expressed as percent of control. Each valueis expressed as the mean±SD, n=3 *p<0.05, **p<0.001 compared to thecontrol.

FIG. 5 shows the effect of A3 adenosine receptor ligands on Aktphosphorylation. Serum starved B16 melanocytes were exposed to 10 μMIB-MECA or MRS-1523 for 10 min, 30 min or 3 h.

FIG. 5A shows western blot analysis of cell protein extracts (20μg/lane) analyzed for Akt phosphorylation compared to total Aktexpression. Time intervals of exposure to the ligands are indicated.

FIG. 5B shows confocal image analysis of B16 melanocytes exposed to theindicated ligands for 3 h, following labeling with anti-phospho-Aktantibody and Cy3-conjugated donkey anti-rabbit antibody, and counterstaining with DAPI. Arrow indicates phospho-Akt. Scale bars correspondto 25μ.

FIG. 6 shows the effect of A3 adenosine receptor ligands on ERK1/2phosphorylation. Serum starved B16 melanocytes were exposed to 10 μMIB-MECA or MRS-1523 for various time periods, and protein extracts ofthe cells were analyzed by western blot analysis (20 μg protein/lane)for ERK phosphorylation and total ERK expression.

FIG. 6A shows analysis of proteins from cells exposed to the indicatedligands for 10 min, 30 min or 3 h.

FIG. 6B shows analysis of proteins from cells exposed to the indicatedligands for 72 h.

FIG. 7 shows Mitf expression and localization in B16 melanocytes treatedwith A3 adenosine receptor ligands for 3 h or 24 h, followed byimmunostaining with anti-Mitf antibody and visualization by confocalmicroscopy. N, nucleus.

FIG. 8 shows the effect of A3 adenosine receptor ligands on skinpigmentation as evaluated by DOPA staining. Samples of human breast skinwere topically treated with A3 adenosine receptor ligands (10 or 50 μM)or with hydroquinone (100 μM) as indicated three times over a cultureperiod of 12 days, during which the medium was replaced every 3 days.

FIG. 8A is a photograph of skin samples following treatment with theindicated compounds after 12 days of culture.

FIG. 8B shows light microscope images (×400) of epidermis peeled fromskin samples treated with IB-MECA or MRS-1523 (10 μM each) orhydroquinone (100 μM) as indicated, stained with DOPA and mounted onslides. FIG. 8D shows light microscope images (×400) of epidermis peeledfrom skin samples treated with IB-MECA or MRS-1523 (50 μM each) orhydroquinone (100 μM) as indicated, stained with DOPA and mounted onslides.

FIGS. 8C and 8E show the degree of pigmentation (density) in the samplesshown in FIGS. 8B and 8D respectively, as evaluated by measurement ofthe light transmission capacity though the peeled epidermis sheets usingsingle channel transmission by light microscopy (×50). D-P-M,DOPA-positive melanocytes.

FIG. 9 shows the effect of A3 adenosine receptor ligands on skinpigmentation as evaluated by Fontana-Masson stain. Formalin fixedparaffin embedded skin sections from samples of human breast skintreated with IB-MECA, MRS-1523 or hydroquinone as described for FIG. 8,were evaluated for Fontana-Masson staining and visualized by lightmicroscopy (×400). IBMX (100 μM) was used as a positive control forinduction of melanogenesis. K, keratinocytes; M, melanocytes.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods as well as pharmaceutical andcosmetic compositions useful for modulating i.e. increasing ordecreasing, the production, secretion and/or accumulation of melanin inskin cells, thereby enabling alteration of pigmentation of the skin andhair. In particular, the present invention provides methods andcompositions comprising use of A3 adenosine receptor agonists forenhancing the tanning process by increasing skin pigmentation, as wellas methods and compositions comprising use of A3 adenosine receptorantagonists for reducing skin pigmentation to lighten the skin color.

The inventors of the present invention have surprisingly found that A3adenosine receptor agonists, such as IB-MECA, cause an increase inmelanin synthesis in melanocytes in a dose-dependent fashion, as shownin Example 1. Furthermore, A3 adenosine receptor agonists cause asignificant increase in melanin secretion from the cells, as shown inExample 3. In addition, agonist treatment further exerts the effect ofaltering accumulation and/or re-distribution of melanosomes within thecell, as shown in Example 3, and moreover may promote melanosometransfer from melanocytes to keratinoctyes, as shown in Example 9.Example 3 further shows that the increase in melanin production occursin a time-dependent fashion, indicating that the invention may beeffectively used for gradually increasing skin pigmentation, which isadvantageous both for cosmetic applications directed at achieving“tanning” effects over a period of time, and for pharmaceuticalapplications directed at ameliorating hypo-pigmentation skin disorders.

The principles of the invention directed to augmenting skin pigmentationhave further been demonstrated in ex vivo studies of skin explants asshown in Examples 8 and 9, which provide confirmation of the increase inmelanin production and/or secretion in melanocytes and/or melaninaccumulation in keratinocytes using a number of histological andmicroscopic methods.

Alternatively, it is now disclosed that A3 adenosine receptorantagonists, such as MRS-1523, cause a decrease in melanin synthesis inmelanocytes in a dose dependent fashion, as shown in Example 2. Thisreduction also occurs in a time-dependent fashion, as shown in Example3. The principles of the invention directed to reducing skinpigmentation have further been demonstrated in ex vivo studies of skinexplants as shown in Examples 8 and 9, which provide confirmation of thedecrease in melanin production and/or secretion in melanocytes and/ormelanin accumulation in keratinocytes using a number of histological andmicroscopic methods.

Examples 5 to 7 suggest that the effect of A3 adenosine receptoragonists in enhancing pigmentation, and the effect of A3 adenosinereceptor antagonists in decreasing pigmentation, may involve Mitfactivation and Mitf downregulation respectively, mediation of whichoccurs via the Akt and ERK signaling pathways.

Definitions

As used herein, the term “ligand of the A3 adenosine receptor” refers toa compound which specifically binds the A3 adenosine receptor andthereby causes either activation of the A3 adenosine receptor (i.e., A3adenosine receptor agonist), or inhibition of the A3 adenosine receptor(i.e., A3 adenosine receptor antagonist).

As used herein, the term “A3 adenosine receptor agonist” refers to aligand of the A3 adenosine receptor, which upon binding to the receptorexerts full or partial activation of that receptor.

As used herein, the term “A3 adenosine receptor antagonist” refers to aligand of the A3 adenosine receptor, which upon binding to the receptorexerts full or partial inhibition of that receptor.

As used herein, the term “selective” means that the binding affinity ofan A3 adenosine receptor agonist or of an A3 adenosine receptorantagonist to the A3 adenosine receptor is at least 20 fold, preferablyat least 50 fold and more preferably at least 100 fold greater than thebinding of the same compound to a heterologous adenosine receptor, forexample the A1 adenosine receptor, the A2a adenosine receptor or the A2badenosine receptor of the same species, or the A3 adenosine receptor ofa different species.

As used herein, the term “allosteric enhancer of the A3 adenosinereceptor” refers to a compound which enhances the binding of an A3adenosine receptor agonist to the A3 adenosine receptor, therebyincreasing the activation effect of the agonist.

As used herein, the term “modulating melanin production” means affectinga change in the pattern of melanin production, for example increasing ordecreasing the overall amount of melanin produced by a cell, orincreasing or decreasing the rate of melanin production by a cell.

As used herein, the term “modulating melanin secretion” means affectinga change in the pattern of melanin secreted from a cell, for exampleincreasing or decreasing the overall amount of melanin secreted from acell, or increasing or decreasing the rate of melanin secreted from acell. The term also encompasses an alteration in the target destinationof melanin secretion such as into the extracellular medium or space, orinto a different cell or into a different cell type.

As used herein, the term “modulating melanin accumulation” meansaffecting a change in the pattern of melanin deposition or sequestrationin or among cell compartments or organelles, or among cell types.

As used herein, the term “skin cell” refers to any type of skin cell,including skin cells involved in melanin production and/or accumulationsuch as melanocytes and keratinocytes, and precursors thereof.

As used herein, the term “at least one skin cell” encompasses both asingle skin cell and a plurality of skin cells, including those formingskin tissue and skin surface areas of any size or dimension, for exampleas measured in square meters of body surface area

A3 Adenosine Receptor Agonists

As used herein, an A3 adenosine receptor agonist (also referred toherein as “A3RAg”) is any compound capable of specifically binding tothe A3 adenosine receptor (also referred to herein as “A3R”), andcapable of fully or partially activating said receptor. The A3RAg isthus a compound that exerts its prime effect though the binding andactivation of the A3R. The ability of a compound to bind A3R may beassessed in a competitive binding assay, typically in which a testcompound is assessed for the ability to displace a radiolabeled form ofa known A3RAg (for example [125I]-AB-MECA) from binding to the A3Rpresent on cells or membranes. Such binding assays are described forexample in Olah et al., Mol Pharmacol. May 1994; 45(5):978-82;Auchambach et al. Mol Pharmacol. November 1997; 52(5):846-60; andKreckler et al., J Pharmacol Exp Ther. April 2006; 317(1):172-80. Theability of a compound to activate A3R may also be assessed in afunctional assay based on determination of downstream signaling events,in particular the effect on adenylyl cyclase as measured by the effect(i.e. increase or decrease) on cAMP level. Such cAMP assays used toassess A3R activation by various compounds are described for example inWan et al., J Pharmacol Exp Ther. January 2008; 324(1):234-43 andAuchambach et al. Mol Pharmacol. November 1997; 52(5): 846-60.

The affinity of an A3RAg to the human A3R as well as its relativeaffinity to the other human adenosine receptors (A1, A2a and A2b) can bedetermined by binding assays and cAMP assays, as described above. Inpreferred embodiments, the A3RAg is a selective A3RAg, meaning that itsbinding affinity for the A3 adenosine receptor is greater than thebinding affinity of the same compound for a different adenosinereceptor, for example the A1 adenosine receptor. In some preferredembodiments, the binding affinity of the A3RAg for the A3 adenosinereceptor is at least 20 times greater than the binding affinity of saidagonist for the A1 adenosine receptor.

In a preferred embodiment, an A3RAg has a binding affinity (K_(i)) forthe human A3 adenosine receptor in the range of less than 200 nM,typically less than 100 nM, preferably less than 50 nM, more preferablyless than 20 nM and even more preferably less than 10 nM. The lower theKi, the lower the dose of the A3RAg that may be used that will beeffective in activating the A3R and thus achieving the desired effect.Thus in certain embodiments, an A3RAg that has a K_(i) for the human A3Rof less than 5 nM and even less than 1 nM may be preferred.

In preferred embodiments, the A3RAg specifically activates the A3R atthe administered levels, and is substantially devoid of activity inactivating any of the other adenosine receptors i.e. the A1, A2a and A2badenosine receptors. In particular, if the administered level of anA3RAg is such that its blood level reaches a level approaching that ofthe K_(i) of the A1, A2a or A2b adenosine receptors, activation of thesereceptors may occur following such administration, in addition toactivation of the A3R An A3RAg is thus preferably administered at a dosesuch that the blood level that will be attained will give rise toessentially only A3R activation.

A3 adenosine receptor agonists which are useful in the present inventioninclude a variety of nucleoside derivatives which are known to act as A3adenosine receptor agonists. As used herein, the term “nucleoside”refers to a compound comprising a sugar backbone, preferably ribose ordeoxyribose, linked to a purine or pyrimidine base by way of N-glycosyllink. The term “nucleoside-derivative” denotes herein a syntheticnucleoside or a nucleoside which underwent chemical modifications by wayof insertion/s, deletion/s or exocyclic and endocyclic substitution/s ofgroup/s therein or conformational modifications which provide aderivative with the desired biological effect, such as enhancing melaninproduction and/or secretion, thereby increasing pigmentation in skin.

In accordance with particular embodiments of the present invention, thedesired biological effect caused by an A3 adenosine receptor agonist isenhancement of melanin synthesis and/or secretion and/or accumulation ina skin cell. A3 adenosine receptor agonists useful for the inventioninclude N⁶-benzyladenosine-5′-uronamide derivatives, especially thosecontaining a methyl- or ethyl-substituted uronamide moiety, which havebeen shown to possess significant A3 adenosine receptor affinity andselectivity. A well known A3 adenosine receptor agonist is N⁶-(3-iodo-benzyl)-adenosine-5′-N-methyl-uronamide (IB-MECA) which isknown to be 50-fold selective for the A3 adenosine receptor versuseither the A1 or A2A receptors.

The A3 adenosine receptor agonist may in particular be anN⁶-substituted-adenosine-5′-uronamide, such as anN⁶-monosubstituted-adenosine-5′-uronamide or anN⁶-disubstituted-adenosine-5′-uronamide.

In some embodiments, the A3 adenosine receptor agonist may be onecategorized as an N⁶-benzyladenosine-5′-uronamide; anN⁶-4-substituted-sulfonamidophenylcarbamoyl-adenosine-5′-uronamide; a2-chloro-N⁶-substituted-4′-thioadenosine-5′-uronamide; or an(N)-methanocarba adenosine-5′-uronamide. Derivatives and analogs ofcompounds in these various classes may also be used.

According to other particular embodiments, the A3 adenosine receptoragonist is selected from the group consisting of N⁶-(3-iodobenzyl)adenosine-5′-(N-methyluronamide) (IB-MECA);2-chloro-N⁶-(3-iodobenzyl)-adenosine-5′-(N-methyluronamide)(Cl-IB-MECA); N⁶-(4-amino-3-iodobenzyl)-adenosine-5′-(N-methyluronamide)(AB-MECA); N⁶-2-(4-aminophenyl) ethyladenosine (APNEA);2-(1-hexynylhexynyl)-N-methyladenosine;2-chloro-N⁶-(3-iodobenzyl)-9-[5-(methylcarbamoyl)-D-ribofuranosyl]adenine(CF-102).

Some other agonistic compounds include(2S,3S,4R,5R)-3-amino-5-[6-(2,5-dichlorobenzylamino)purin-9-yl]-4-hydroxytetrahydrofuran-2-carboxylicacid methyl amide (CP-532,903); and ring-constrained(N)-methanocarba-5′-uronamide 2,N⁶-disubstituted adenine nucleosidessuch as(1′R,2′R,3′S,4′R,5′S)-4-{2-chloro-6-[(3-chlorophenylmethyl)amino]purin-9-yl}-1-(methylaminocarbonyl)bicyclo[3.1.0]hexane-2,3-diol(MRS-3558; Tchilibon et al., 2005, J. Med. Chem., 48:1745-58; Melman etal Bioorg Med Chem Lett May 1, 2008; 18(9):2813-2819; Tosh et al.,Bioorg Med Chem Jan. 15, 2010; 18(2):508-17).

In a particular embodiment, the A3 adenosine receptor agonist isselected from the group consisting of N⁶-(3-iodobenzyl)adenosine-5′-(N-methyluronamide) (IB-MECA);2-chloro-N⁶-(3-iodobenzyl)-adenosine-5′-(N-methyluronamide)(Cl-IB-MECA); 2-(1-hexynylhexynyl)-N-methyladenosine;2-chloro-N⁶-(3-iodobenzyl)-9-[5-(methylcarbamoyl)-D-ribofuranosyl]adenine(CF-102); and(2S,3S,4R,5R)-3-amino-5-[6-2,5-dichlorobenzylamino)purin-9-yl]-4-hydroxytetrahydrofuran-2-carboxylicacid methyl amide (CP-532,903).

Selective A3 adenosine receptor agonists include thionucleoside analogsof IB-MECA, such as those described in WO 2004/038006 and U.S. Pat. No.7,199,127, including for example(2R,3S,4R)-2-[2-chloro-6-(3-iodobenzylamino)purin-9-yl]-3,4-dihydroxytetrahydrothiophene-2-carboxylicacid methyl amide;(2S,3S,4R,5R)-5-(2-chloro-6-methylaminopurin-9-yl)-3,4-dihydroxytetrahydrothiophene-2-carboxylicacid methyl amide; and(2S,3S,4R,5R)[5-(2-chloro-6-(3-iodobenzylamino)purin-9-yl)-3,4-dihydroxytetrahydrothiophene-2-carboxylicacid methyl amide.

Selective A3 adenosine receptor agonists further include thioadenosineanalogs of Cl-IB-MECA, as disclosed for example in Jeong et al., J MedChem 2003: 46:3775-3777; Jeong et al., J Med Chem 2006: 49:273-281; andChoi et al., Bioorg Med Chem Dec. 1, 2009; 17(23):8003-11.

A3 adenosine receptor agonists comprising a bicyclic ring substituentare described in U.S. Pat. No. 7,414,036, and include for example,N⁶-(4-iodo-2-picolyl)-adenosine-5′-N-methyluronamide;N⁶-(4-methyl-2-picolyl)-adenosine-5′-N-methyluronamide;N⁶-(2-picolyl)-adenosine-5′-N-methyluronamide;N⁶-(6-acetyl-2-picolyl)-adenosine-5′-N-methyluronamide;N⁶-(4-iodo-2-picolyl)-2-(2-phenyl-1-ethynyl)-adenosine-5′-N-methyluronamide;andN⁶-[(2-dimethylamino-7-iodo-1,3-benzoxazol-5-yl)-methyl]-adenosine-5′-N-methyluronamide.

N⁶-4-sulfonamido-adenosine-5′-uronamide derivatives which are selectiveA3 adenosine receptor agonists are disclosed in U.S. Pat. No. 7,511,133and in Baraldi et al., J. Med. Chem. Oct. 21, 2004; 47(22):5535-40, andinclude for example,1-deoxy-1-[6-[[[[4-[N-(allyl)-N(methyl)aminosulfonyl]phenyl]amino]carbonyl]amino]-9H-purin-9-yl]-N-ethyl-β-D-ribofuranuronamide;1-deoxy-1-[6-[[[(4-(N-methyl-N-isopropyl-amino-sulfonyl)-phenyl)-amino]carbonyl]amino]-9H-purin-9-yl]-N-ethyl-β-D-ribofuranuronamide;1-deoxy-1-[6-[[[[4-[N,N-(dimethyeamino-sulfonyl]phenyl]-amino]carbonyl]amino]-9H-purin-9-yl]-N-ethyl-β-D-ribofuranuronamide;and1-deoxy-1-[6-[[[[4-[N,N-bis(allyl)amino-sulfonyl]phenyl]amino]carbonyl]amino]-9H-purin-9-yl]-N-ethyl-β-D-ribofuranuronamide;1-deoxy-1-[6-[[[[4-[N,N-bis(ethyl)amino-sulfonyl]phenyl]amino]carbonyl]amino]-9H-purin-9-yl]-N-ethyl-β-D-ribofuranuronamide.

Various other A3 adenosine receptor agonists may be used in theinvention, such as those disclosed for example in U.S. Pat. Nos.5,688,774; 5,773,423; 5,573,772; 5,443,836; 6,048,865; 6,177,444;6,194,449; 6,323,214; 7,199,127; 7,414,036; 7,465,715; 7,511,133; U.S.Patent Application No. 2005/0250729; WO 95/02604; WO 99/20284; WO99/06053; WO 97/27173; WO 01/23399; WO 02/055085; WO 02/070532; and WO2004/038006.

A3 Adenosine Receptor Antagonists

As used herein, an A3 adenosine receptor antagonist is any compoundcapable of specifically binding to the A3 adenosine receptor (A3R), andcapable of fully or partially inhibiting i.e. inactivating saidreceptor. The A3 adenosine receptor antagonist is thus a compound thatexerts its prime effect though the binding and inactivation of the A3R.Binding and functional assays that may be used for the characterizationof such antagonists are essentially those that are used for thecharacterization of A3 adenosine receptor agonists, as described herein.

In particular embodiments, the A3 adenosine receptor antagonist has abinding affinity (Ki) for the A3 adenosine receptor that is less thanabout 200 nM, preferably less than about 100 nM, more preferably lessthan about 50 nM, even more preferably less than 10 nM. In otherembodiments, the binding affinity of the A3 adenosine receptorantagonist for the adenosine A3 receptor is at least 20 times greaterthan the binding affinity of said antagonist for the A1 adenosinereceptor.

A3 adenosine receptor antagonists which are useful in the presentinvention include a variety of known A3 adenosine receptor antagonistsselected from but not limited to compounds variously classified asxanthines and derivatives thereof (as disclosed for example in Priego etal., J Med Chem. Aug. 1, 2002; 45(16):3337-44; and Muller et al., J MedChem. Aug. 1, 2002; 45(16):3440-50; dihydropyridines and derivativesthereof (as disclosed for example in Jiang et al., 1997, J. Med. Chem.40:2596-608 and U.S. Pat. No. 6,376,521); pyridines and derivativesthereof (as disclosed for example in Li et al., 1999, J. Med. Chem.42:706-21); flavonoids and derivatives thereof (as disclosed for examplein Ji et al., 1996, J. Med. Chem., 39:781-8); isoquinolines andderivatives thereof (as disclosed for example in van Muijlwijk-Koezen etal., J Med Chem. Jun. 1, 2000; 43(11):2227-38; and Gao et al., MolPharmacol. November 2001; 60(5):1057-63); triazoloquinazolines andderivatives thereof (as disclosed for example in Kim et al., J. MedChem. Oct. 11, 1996; 39(21):4142-8); triazolonaphthiridines andderivatives thereof, thiazolopyrimidines and derivatives thereof,2-arylpyrazolo[3,4-c]quinoline derivatives,5-N-(phenylcarbamoyl)amino-8-2-(2-furly)pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidinederivatives (as disclosed for example in Baraldi and Borea, 2000, TiPS,21:456-9); imidazoquinolines and derivatives thereof (as disclosed forexample in U.S. Patent Application No. 2008/0255110); triazolopurinesand derivatives thereof (as disclosed for example in U.S. Pat. No.6,288,070); deazapurine derivatives (as disclosed for example in U.S.Pat. No. 7,504,407); and pyrazolo-triazolo-pyrimidine,triazolo-triazolo-pyrimidines and imidazolo-triazolo-pyrimidines andderivatives thereof (as disclosed for example in U.S. Pat. Nos.7,470,698 and 6,448,253).

Particular examples of A3 adenosine receptor antagonists include3-propyl-6-ethyl-5[(ethylthio)carbonyl]-2-phenyl-4-propyl-3-pyridinecarboxylate (MRS-1523);1,4-dihydro-2-methyl-6-phenyl-4-(phenylethynyl)-3,5-pyridinedicarboxylicacid 3-ethyl-5-[(3-nitrophenyl)methyl]ester (MRS-1334);3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(±)-dihydropyridine-3,5dicarboxylate (MRS-1191); 3-ethyl5-benzyl-2-methyl-6-phenyl-4-styryl-1,4-(±)-dihydropyridine-3,5-dicarboxylate(MRS-1097);5-n-butyl-8-(4-trifluoromethylphenyl)-3H-[1,2,4]triazolo-[5,1-i]purine(OT-7999);(2R,3R,4S,5S)-2-[N⁶-3-iodobenzyl)adenos-9′-yl]-7-aza-1-oxa-6-oxospiro[4,4]-nonan-4,5-diol(MRS-1292); N-(2-methoxyphenyl)-N′-[2-(3-pyridinyl)-4-quinazolinyl]-urea(VUF-5574);(8R)-8-ethyl-1,4,7,8-tetrahydro-4-5H-imidazo[2,1-i]purin-5-one (PSB-11);2-phenoxy-6-(cyclohexylamino)purine (MRS-3777);5N-(4-methoxyphenylcarbamoyl)amino-8-propyl-2-(2-furyl)pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine(MRE3008F20) and derivatives and analogs thereof.

In a particular embodiment, the A3 adenosine receptor antagonist isselected from the group consisting of3-propyl-6-ethyl-5[(ethylthio)carbonyl]-2-phenyl-4-propyl-3-pyridinecarboxylate (MRS-1523);1,4-dihydro-2-methyl-6-phenyl-4-(phenylethynyl)-3,5-pyridinedicarboxylicacid 3-ethyl-5-[(3-nitrophenyl)methyl]ester (MRS-1334); and3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(±)-dihydropyridine-3,5dicarboxylate (MRS-1191).

Suitable antagonists further include those disclosed in Jacobson et al.,1997, Neuropharmacology, 36:1157-65; Yao et al., 1997, Biochem. Biophys.Res. Commun., 232:317-22; Kim et al., 1996, J. Med. Chem., 39:4142-48;van Rhee et al., 1996, Drug Devel. Res., 37:131; van Rhee et al., 1996,J. Med. Chem., 39:2980-9; Siquidi et al., 1996, Nucleosides, Nucleotides15:693-718; van Rhee et al., 1996, J. Med. Chem., 39:398-406; Jacobsonet al., 1995, Drugs of the Future, 20:689-699; Jacobson et al., 1995, J.Med. Chem., 38:1720-35; Karton et al., 1996, J. Med. Chem.,39:2293-2301; Kohno et al., 1996, Blood, 88:3569-74; Jiang et al., 1996,J. Med. Chem., 39:4667-75; Yao et al., 1997, Biochem. Biophys. Res.Commun. 232:317-22; and Jiang et al., 1996, J. Med. Chem. 40:2596-2608;Jeong L. S., 2008, Nucleic Acids Sympo. Series., 52: 79-80, Choi et al.,Nucleic Acids Synp Ser (Oxf). 2008; (52):645-646; U.S. Pat. No.7,435,740 and U.S. Patent Applications Nos. 2004/0067932 and2006/0178385.

Allosteric Enhancers

The compositions and methods of the invention for increasing melaninproduction may further comprise the use of an allosteric enhancer of theA3 adenosine receptor. Such allosteric enhancers are disclosed forexample in Gao et al., Mol Pharmacol. July 2002; 62(1):81-9; Gao et al.,Mol Pharmacol. November 2001; 60(5):1057-63; and Goblyos et al., J MedChem. Jun. 1, 2006;49(11):3354-61.

Specific examples of allosteric enhancers of the A3 adenosine receptorinclude, but are not limited to, 1H-imidazo[4,5-c]quinolin-4-amines,3-(2-pyridinyl)isoquinolines and 2,4-disubstituted quinolones. Forexample 1H-imidazo[4,5-c]quinolin-4-amine includeN-(3,4-dichloro-phenyl)-2-cyclohexyl-1H-imidazo[4,5-c]quinolin-4-amine(LUF-6000); N-phenyl-2-cyclopentyl-1H-imidazo[4,5-c]quinolin-4-amine(DU124183). The 3-(2-pyridinyl)isoquinoline may be selected from4-methoxy-N-[7-methyl-3-(2-pyridinyl)-1-isoquinolinyl]benzamide(VUF5455); 4-methyl-N-[3-(2-pyridinyl)-1-isoquinolinyl]benzamide(VUF8502); 4-methoxy-N-[3-(2-pyridinyl)-1-isoquinolinyl]benzamide(VUF8504); and N-[3-(2-pyridinyl)-1-isoquinolinyl]benzamide (VUF8507).The 2,4-disubstituted quinoline may be selected fromN-(2-anilinoquinolin-4-yl)cyclopentanecarboxamide;N-{2-[(3,4-dichlorophenyl)amino]quinolin-4-yl}cyclopentanecarboxamide;N-[2-(benzylamino)quinolin-4-yl]cyclopentanecarboxamide;N-{2-[(4-methylphenyl)amino]quinolin-4-yl}cyclopentanecarboxamide;N-[2-(2,3-dihydro-1H-inden-5-ylamino)quinolin-4-yl]cyclopentanecarboxamide;N-{2-[(4-methoxyphenyl)amino]quinolin-4-yl}cyclopentanecarboxamide;N-{2-[(4-chlorophenyl)amino]quinolin-4-yl}cyclopentanecarboxamide;N-[2-(cyclopentylamino)quinolin-4-yl]cyclopentanecarboxamide;N-[2-(1H-indazol-6-ylamino)quinolin-4-yl]cyclopentanecarboxamide;N-(2-anilinoquinolm-4-yl)cyclohexanecarboxamide;N-{2-[(3,4-dichlorophenyl)amino]quinolin-4-yl}cyclohexanecarboxamide;N-{2-[(4-methylphenyl)amino]quinolin-4-yl}cyclohexanecarboxamideN-[2-(2,3-dihydro-1H-inden-5-ylamino)quinolin-4-yl]cyclohexanecarboxamide;N-(2-anilinoquinolin-4-yl)benzamide;N-{2-[(3,4-dichloro-phenyl)amino]quinolin-4-yl}benzamide;N-(2-anilinoquinolin-4-yl)-2-furamide;N-{2-[(3,4-dichlorophenyl)amino]quinolin-4-yl}-2-furamide;N-(2-anilinoquinolin-4-yl)cyclobutanecarboxamide; andN-{2-[(3,4-dichlorophenyl)amino]quinolin-4-yl}cyclobutanecarboxamide.

Adjunct Ingredients

The skin lightening compositions of the invention may further contain atleast one other adjunct ingredient in addition to the specific A3adenosine receptor antagonist. Adjunct ingredients include, but are notlimited to, hydroxytetronic acid and/or hydroxytetronic acidderivatives, tetronic acid and/or tetronic acid derivatives,hydroquinone, α-hydroxy acids, and fatty acid esters of ascorbic acid.According to some embodiments some whitening compositions of theinvention employ more than one adjunct ingredient.

Especially preferred whitening compositions of the invention thatcontain an adjunct ingredient employ either a hydroxytetronic acidand/or hydroxytetronic acid derivatives as those described in U.S. Pat.No. 7,019,029 or tetronic acid (2,4-furandione, formula C₄H₆O₅), or atetronic acid derivative, or hydroquinone (sometimes also calledp-dihydroxybenzene or 1,4 benzenediol), or both, in addition to thehydroxytetronic active ingredient in the formulation. Tetronic acid andits derivatives have been suggested to be useful in the suppression ofmelanin production by inhibiting tyrosinase and tyrosine phosphatases(Sodeoka, M., 2001, J. Med. Chem. 44:3216-22), which can augment theoverall skin whitening observed when compositions of the invention areapplied to skin, as does hydroquinone. Preferred tetronic acid adjunctingredients are tetronic acid derivatives that inhibit tyrosinase ortyrosine phosphatase. These typically have an acyl or other hydrophobicgroup in the 3-position and a free tetronic acid moiety such as5-(4-benzoylbenzoyl)oxymethyl-3-hexadecanoyltetronic acid reported bySodeoka, et al., cited above. Other non-limiting examples include3-hexadecanoyl-5-methanesulfonyloxymethyltetronic acid,3-hexadecanoyl-5-methyltetronic acid, and 3-hexadecanoyltetronic acid.Typical tetronic acid or tetronic acid derivative and/or hydroquinoneconcentrations range from about 0.25% to about 25% by weight, morenarrowly from about 1% to about 5%, and even more narrowly from about 2%to about 4% by weight.

As used herein, the term “α-hydroxy acid” has reference to andencompasses the general class of organic compounds containing at leastone hydroxy group and at least one carboxyl group, and wherein at leastone hydroxyl group is located on the α-carbon atom. Typically, thecompounds are organic acids having at least one carboxylic acid groupand at least one hydroxyl group on the α-carbon atom, and may containother functional groups including additional hydroxyl and carboxylicacid moieties. Preferred α-hydroxy acids and/or α-hydroxy acidderivatives are those which are less bulky structurally, typicallyhaving a one- to three-carbon backbone, so that they penetrate the skinwell such as those disclosed in U.S. Pat. No. 5,965,618. Where employed,glycolic and/or lactic acid or their derivatives are preferred. Lacticacid was suggested as a skin-whitening agent in U.S. Pat. No. 5,262,153.Typical hydroxy acid concentrations range from about 1% to about 25% byweight, more specifically from about 2% to about 15%, and even morespecifically from about 3% to 10% by weight. Typical hydroxytetronicacid concentrations range from about 8% to 12% by weight; morespecifically from about 3% to about 7% by weight.

Fat-soluble fatty acid esters of ascorbic acid (vitamin C) are employedas alternate or additional adjunct ingredients in other embodiments,alone or in combination with hydroquinone or α-hydroxy acids. The moreoxidation-resistant saturated fatty acid esters of ascorbic acid arepreferred, including, but not limited to, ascorbyl laurate, ascorbylmyristate, ascorbyl palmitate, ascorbyl stearate, and ascorbyl behenate.Ascorbyl palmitate is used in one embodiment. The esters may be preparedusing hydrogenated oils or fats, or fractions thereof. Ascorbyl stearateprepared using canola oil, for example, commonly contain about 4%ascorbyl palmitate. It is an advantage of the invention that where fattyacid esters of ascorbic acid are employed as an adjunct ingredient, theyhelp provide emollient properties to the composition. Typicalconcentration ranges of ascorbyl palmitate vary from about 0.25% toabout 10%, more narrowly from about 2% to about 8%, and even morenarrowly from about 3% to about 5% by weight.

Therapeutic Uses, Administration and Formulations

The invention encompasses therapeutic uses of A3 adenosine receptorantagonists for treating and ameliorating skin hyper-pigmentationdisorders and conditions, as well as therapeutic uses of A3 adenosinereceptor agonists for treating and ameliorating skin hypo-pigmentationdisorders and conditions. Hyper-pigmentation conditions which may betreated with the invention include for example, pigmented spots, lentigosenilis, freckles, café au lait spots, liver spots, ephelides,periorbital darkening, post-inflammatory hyper-pigmentation, pigmentedkeratosis, melasma and chloasma.

Hypo-pigmentation conditions which may be treated with the inventioninclude for example, pigmented vitiligo, piebaldism, leukoderma due tocicatrisation, and nevus depigmentosis.

These and other abnormal or undesirable skin pigmentation conditions arewell understood by those of skill in the art, and are described forexample in: Rose, Pigmentary disorders. Med. Clin North Am. November2009; 93(6):1225-39; Tones et al., Melasma and other disorders ofhyperpigmentation. In: Cutaneous Medicine and Surgery. Arndt K A, LeBoitP E, Robinson J K, and Wintroub B U, eds. W.B. Saunders Co:Philadelphia, 1996, pp. 1233-1241; Young et al., Melasma update. ActasDermosifiliogr. December 2009; 100 Suppl 2:110-3; Taylor et al.,Postinflammatory hyperpigmentation. J Cutan Med Surg. July-August 2009;13(4):183-91; Hershkovitz et al., Monogenic pigmentary skin disorders:genetics and pathophysiology. Isr Med Assoc J. October 2008;10(10):713-7; Ramos-e-Silva et al., Hair, nail, and pigment changes inmajor systemic disease. Clin Dermatol. May-June 2008; 26(3):296-305; Yuet al., Diagnosis and treatment of pigmentary disorders in Asian skin.Facial Plast Surg Clin North Am. August 2007; 15(3):367-80, vii;Dessinioti et al., A review of genetic disorders of hypopigmentation:lessons learned from the biology of melanocytes. Exp Dermatol. September2009; 18(9):741-9.

A preferred subject to be treated is a human.

Further encompassed within the invention are cosmetic uses of A3adenosine receptor antagonists for lightening dark skin, for example inindividuals of population groups having genetically dark skin. Furtherencompassed within the invention are cosmetic uses of A3 adenosinereceptor agonists for producing a tanned skin effect in individualshaving fair skin.

Further encompassed within the invention is the use of A3 adenosinereceptor agonists for treating depigmentation of a skin site whichoccurs secondary to surgical skin graft procedures, and the use of A3adenosine receptor antagonists for treating hyperpigmentation of a skinsite which occurs secondary to surgical skin graft procedures. It iswell known in the art that skin graft procedures may result indepigmentation or hyper-pigmentation at the graft site. Such anundesirable outcome may occur following cosmetic surgery, burntreatment, in particular split-thickness grafts or any other medicalintervention requiring a skin graft procedure.

The cosmetic and pharmaceutical compositions comprising as and activeingredient an A3 adenosine receptor agonist or an A3 adenosine receptorantagonist may be administered to a subject by any route, includeing butare not limited to, direct administration into, onto or in the vicinityof hyper-pigmented or hypo-pigmented tissue, topical, intradermal,transdermal, subcutaneous, parenteral (including intravenous,intraarterial, intramuscular, intraperitoneal administration), as wellas intranasal and oral administration. The compounds can be administeredby any convenient route and can be administered together with othertherapeutically active agents, such as an allosteric enhancer of the A3adenosine receptor or an adjunct skin lightening or whitening agent asdescribed herein. According to some embodiments, the route ofadministration is a route which enables the active ingredient i.e. A3adenosine receptor agonist or A3 adenosine receptor antagonist to reachthe blood stream, suitably intramuscular, intraperitoneal, intravenous,subcutaneous, intranasal or oral administration.

It may be desirable to administer the cosmetic or pharmaceuticalcomposition of the invention locally to the hyper-pigmented orhypo-pigmented tissue in need of treatment; this can be achieved by, forexample, and not by way of limitation, local injection or topicalapplication, e.g., in conjunction with a wound dressing.

The terms “effective amount” and “amount effective” are usedinterchangeably herein to refer to the amount and/or dose of an A3adenosine receptor ligand which achieves its intended purpose. In thecase where increasing any of melanin production, secretion oraccumulation is desired, for example for treating a hypo-pigmentationcondition, an effective amount of an A3 adenosine receptor agonist isthat which results in a measurable or detectable increase in melaninlevel or skin pigmentation or relevant change in melanin location withinor among cells. Similarly, in the case where decreasing any of melaninproduction, secretion or accumulation is desired, for example fortreating a hyper-pigmentation condition, an effective amount of an A3adenosine receptor antagonist is that which results in a measurable ordetectable decrease in melanin level or skin pigmentation or relevantchange in melanin location within or among cells.

An example of an effective amount is a daily administration of an A3adenosine receptor agonist within the range of between about 1 μg/kgbody weight and about 10 mg/kg body weight. Such an amount of an A3adenosine receptor agonist is typically administered in a single dailydose although at times a daily dose may be divided into several dosesadministered throughout the day or at times several daily doses may becombined into a single dose to be given to the patient once everyseveral days, particularly if administered in a sustained or controlledrelease formulation.

Administration of the A3 adenosine receptor agonist or the A3 adenosinereceptor may be carried out over a period of at least two weeks, or atleast one month, or at least two months, or longer, in order to achievethe desired outcome.

The pharmaceutical compositions of the present invention may contain,for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of activeingredient in combination with a pharmaceutically acceptable carrier.

As used herein, the term “pharmaceutically acceptable carrier” means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting a compound(s) of thepresent invention within or to the subject such that it can performs itsintended function. A carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the patient. Some examples of materials which can serve aspharmaceutically acceptable carriers include: sugars, such as lactose,glucose and sucrose; starches, such as corn starch and potato starch;cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; powdered tragacanth; malt;gelatin; talc; excipients, such as cocoa butter and suppository waxes;oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; glycols, such as propylene glycol;polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;esters, such as ethyl oleate and ethyl laurate; agar; buffering agents,such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol;phosphate buffer solutions; and other non-toxic compatible substancesemployed in pharmaceutical formulations.

According to some embodiments, the compositions of the invention can beapplied to the skin. According to some other embodiments, thecompositions of the invention can be applied to hair follicles. Thecompositions may be in the form of, for example, and not by way oflimitation, an ointment, cream, gel, paste, foam, aerosol, pad or gelledstick.

A3 adenosine receptor agonists and antagonists according to theprinciples of the invention can be delivered in a controlled releasesystem. In one embodiment, the A3 Adenosine receptor agonists andantagonists of the invention can be administered in combination with abiodegradable, biocompatible polymeric implant, which releases the A3Adenosine receptor antagonist or agonist over a controlled period oftime at the hyper-pigmented or hypo-pigmented tissue respectively.Examples of preferred polymeric materials include polyanhydrides,polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinylacetate, copolymers and blends thereof (see Medical applications ofcontrolled release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton,Fla.).

According to some preferred embodiments, the A3 adenosine receptoragonist or antagonist is administered as part of a topical formulation.The compositions of the present invention formulated for topicaladministration comprise the melanin modulating agent in an effectivedose together with one or more pharmaceutically or therapeuticallyacceptable carriers and optionally other ingredients. Variousconsiderations are described, e.g., in Gilman et al. (eds) (1990) ThePharmacological Bases Of Therapeutics, 8th Ed., Pergamon Press; NovelDrug Delivery Systems, 2nd Ed., Norris (ed.) Marcel Dekker Inc. (1989),and Remington's Pharmaceutical Sciences,.

Typically, the formulations will comprise a preparation for delivering amelanin modulating agent directly to the skin or hair comprising themodulating agent, typically in concentrations in the range from about0.001% to 20%; preferably, from about 0.01 to about 10%; morepreferably, from about 0.1 to about 5%; together with a non-toxic,pharmaceutically acceptable topical carrier. Topical preparations can beprepared by combining the melanin modulating agent with conventionalpharmaceutical diluents and carriers commonly used in topical dry,liquid, cream and aerosol formulations. Typically, the preparation ofsuitable dosage forms (e.g., sprays, ointments, pastes, creams, lotions,gels, and solutions) will comprise mixing the active compound understerile conditions with diluents and carriers along with anypreservatives, buffers, or propellants which may be required.

Ointment and creams may, for example, be formulated with an aqueous oroily base with the addition of suitable thickening and/or gellingagents. Such bases may include water and/or an oil such as liquidparaffin or a vegetable oil such as peanut oil or castor oil. Thickeningagents which may be used according to the nature of the base includesoft paraffin, aluminum stearate, cetostearyl alcohol, propylene glycol,polyethylene glycols, woolfat, hydrogenated lanolin, beeswax, and thelike.

Lotions may be formulated with an aqueous or oily base and will, ingeneral, also include one or more of the following: stabilizing agents,emulsifying agents, dispersing agents, suspending agents, thickeningagents, coloring agents, perfumes, and the like.

Drops may be formulated with an aqueous base or non-aqueous base alsocomprising one or more dispersing agents, suspending agents,solubilizing agents, and the like.

The topical pharmaceutical compositions according to this invention mayalso include one or more preservatives or bacteriostatic agents, e.g.,methyl hydroxybenzoate, propyl hydroxybenzoate, chlorocresol,benzalkonium chlorides, and the like. The topical pharmaceuticalcompositions also can contain other active ingredients such asantimicrobial agents, particularly antibiotics, anesthetics, analgesics,and antipruritic agents.

In some embodiments, active and/or adjunct ingredients are added to asunscreen or sunblock formulations so that topical application has thefurther advantage of preventing repigmentation during and/or aftertreatment. Preferred formulae of this type are SPF 15 or higher. Many ofthese preferred embodiments contain titanium dioxide or zinc oxide whichadditionally smooth and lubricate the skin and help minimize sideeffects in sensitive skin.

Parenterally administered formulations are generally prepared in a unitdosage injectable form (solution, suspension, emulsion). Apharmaceutical formulation suitable for injection includes sterileaqueous solutions or dispersions and sterile powders for reconstitutioninto sterile injectable solutions or dispersions. The carrier employedcan be a solvent or dispersing medium containing, for example, water,ethanol, polyol (for example, glycerol, propylene glycol, lipidpolyethylene glycol and the like), suitable mixtures thereof andvegetable oils. Non-aqueous vehicles such as cottonseed oil, sesame oil,olive oil, soybean oil, corn oil, sunflower oil, or peanut oil andester, such as isopropyl myristate, may also at times be used as solventsystems for the active ingredient.

Additionally, various additives which enhance the stability, sterilityand isotonicity of the compositions, including antimicrobialpreservatives, antioxidants, chelating agents and buffers can be added.Prevention of microbial growth can be ensured by inclusion of variouspreservatives, for example, parabens, chlorobutanol, phenol, sorbic acidand the like.

For the purpose of oral administration, the active ingredient may beformulated in the form of tablets, suspensions, solutions, emulsions,capsules, powders, syrups and the like.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules and the like), the active ingredient is mixedwith one or more pharmaceutically acceptable carriers, such as sodiumcitrate or dicalcium phosphate, and/or any of the following: fillers orextenders, such as starches, lactose, sucrose, glucose, mannitol, and/orsilicic acid; binders, such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia;humectants, such as glycerol; disintegrating agents, such as agar-agar,calcium carbonate, potato or tapioca starch, alginic acid, certainsilicates, and sodium carbonate; solution retarding agents, such asparaffin; absorption accelerators, such as quaternary ammoniumcompounds; wetting agents, such as, for example, cetyl alcohol andglycerol monostearate; absorbents, such as kaolin and bentonite clay;lubricants, such a talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; andcoloring agents. In the case of capsules, tablets and pills, thepharmaceutical compositions may also comprise buffering agents. Solidcompositions of a similar type may also be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugars, as well as high molecular weight polyethylene glycols andthe like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

The term “ameliorate” is used here in its broadest sense to refer to thereduction of the pigmentation in patients suffering fromhyper-pigmentation. Alternatively, it refers to an increase inpigmentation in patient suffering from hypo-pigmentation. The termincludes any of the arrest, prevention, decrease, and improvement in anyof the symptoms of hyper-pigmentation and hypo-pigmentation, bothtemporary and long term. Amelioration of hyper-pigmentation orhypo-pigmentation is a further example of successful treatment ortherapy.

The following examples are presented in order to more fully illustratecertain embodiments of the invention. They should in no way, however, beconstrued as limiting the broad scope of the invention. One skilled inthe art can readily devise many variations and modifications of theprinciples disclosed herein without departing from the scope of theinvention.

EXAMPLES

Materials and Methods used for the Examples disclosed herein are asfollows:

Reagents. IB-MECA, MRS-1523 and IBMX were purchased from Sigma. For eachreagent, a stock solution of 10 mM was prepared in dimethyl sulfoxide(DMSO), and further dilutions in DMEM medium (Dulbecco's modifiedEagle's medium) were prepared. DMEM media, fetal calf serum, andantibiotics for cell cultures were obtained from Kibbutz Beit-Haemek,Israel. Kojic acid, synthetic melanin and anti-phospho-ERK antibody werepurchased from Sigma. Anti-ERK-2 antibody and anti-phospho-ERK antibodywere purchased from Santa Cruz Biotechnology. Anti-Akt-1 antibody andanti-phospho-Akt antibody were purchased from Cell Signaling, andanti-Mitf antibody was purchased from Abcam. BCA™ Protein Assay Kit andECL Kit were purchased from Thermo Scientific.

Cell cultures. B16 F1 melanoma cells were grown adherently andmaintained in DMEM (without phenol red), containing 10% fetal calfserum, 1% antibiotics (penicillin, streptomycin and nystatin, each at100 U/ml), and 2 mM L-glutamine. Cells were cultured at 37° C. in 5%CO₂, and passaged two to thee times weekly at a ratio of 1:10.

Melanin assay. Melanin content was determined as reported by Sato etal., (2008), Biol. Pharm. Bull., 31(1):33-7, with modifications. Cells(6.5×10⁴) were seeded into 6 well plates with 5 ml of DMEM medium(described above) and incubated at 37° C. with 5% CO₂ for 3-4 hours toenable cell adherence. Cells were then exposed to test chemicals(IB-MECA, MRS-1523 or kojic acid) at the desired concentration andincubation periods. After the treatment, the supernatants were collectedto determine the amount of melanin secreted to the medium. Cells werewashed with PBS, detached by incubation with trypsin/EDTA and then DMEMwith serum (0.75 ml) was added to each well. Cells were collected bycentrifugation and lysed by exposure to 2N NaOH (1 ml) at 60° C. for 1 hand the melanin content was determined by measuring the absorption at400 nm. The amount of protein in each lysate was determined using bovineserum albumin is used as the standard. The amount of melanin in eachsample was calculated using synthetic melanin as standard. The resultswere expressed as the melanin content per mg protein (μg/mg protein). Tocompare among treatments, the results were expressed as percent relativeto control.

Western blot analysis. B16 melanocytes grown in 6-well plates were serumstarved overnight and then incubated in the presence of IB-MECA orMRS-1523 (10 μM), for different time intervals at 37° C. Cells were thenrinsed with ice cold PBS and lysed by addition of RIPA buffercontaining: proteinase inhibitor (PIC, 1:50 v/v), 0.1 mMphenylmethylsulphonylfluoride (PMSF) and 1 mM sodium orthovandate. Cellsdebris was removed by centrifugation. The protein level in thesupernatants was determined using BSA as standard. Equal amounts of thesample (20 μg) were separated by SDS-PAGE using 10% polyacrylamide gels.The resolved proteins were then electroblotted onto nitrocellulosemembrane. Membranes were blocked with 1% bovine serum albumin andincubated with anti-ERK-2 antibody (1:1000) or anti-phospho-ERK antibody(1:10000) for 1 h at room temperature, or with anti-Akt-1 antibody(1:1000) or with anti-phospho-Akt antibody (1:000) at 4° C. for 24 h.Blots were then washed and incubated with horseradish peroxidase(conjugated secondary antibodies), for 1 hour at room temperature. Theproteins were detected using ECL kit. For the detection of ERKphosphorylation after 72 hours of exposure to the ligands, non-starvedcells were used (with 10% FCS).

Immunostaining and confocal microscopy. B16 melanocytes were grown for24 h on glass cover-slips coated with poly-L-lysine (500 ug/ml) in 6wells plates in the presence of IB-MECA or MRS-1523 (10 μM), for 3 h or24 h at 37° C. Cells were fixed in 4% formaldehyde in phosphate bufferedsaline (PBS) for 1 h at room temperature. The fixed cells were rinsedthee times for 1 min with PBS. To block non-specific interaction of theantibodies, cells were incubated with PBS (1% BSA, 0.1% Triton X-100).For staining with anti-phospho-Akt antibody, cells were incubated withthe primary antibody at a dilution of 1:1000 for 24 h at 4° C. Cellswere washed thee times for 3 min with PBS and incubated withCy3-conjugated donkey anti-rabbit antibody at a dilution of 1:250 in PBSfor 2 h in the dark. Cells were washed thee times with PBS and mountedwith Fluoromount (Sigma). Cells were visualized by confocal microscopy(excitation 553 and emission at 568, Lieca).

For staining with anti-Mitf antibody, cells were treated as describedabove and incubated with primary anti-Mitf antibody at a dilution of1:100 for 24 h at 4° C. and with the secondary antibody Alexa Fluor®488-conjugated donkey anti-mouse antibody at a dilution of 1:250 for 2 hin the dark.

Skin organ culture. Samples of human breast skin obtained during breastsurgery were cut into pieces (0.5×0.5 cm) and cultured in DMEM medium(described above) in 6 well plates and incubated at 37° C. with 5% CO₂for 12 days. Skin samples were topically treated with 10 or 50 μM ofvarious A3 adenosine receptor ligands or 100 μM hydroquinone thee timesduring the incubation period and the medium was exchanged every 3 days.

DOPA staining. Treated skin samples were washed with PBS and theepidermal sheets were separated from the dermis and incubated in 0.1%L-3,4-DOPA in PBS for 4 h at 37° C. The epidermis sheets were then fixedwith 4% formaldehyde solution, dehydrated by graded alcohol, cleared inxylene, and mounted on glass slides.

Skin melanin evaluation. DOPA positive melanocytes were visualized bylight microscopy (×400 magnification). Light transmission capacity ofthe epidermis sheet was determined by taking 3 images of each sheet fromdifferent zones of the epidermis of each treatment (×50 magnification).Light transmission of each area in the epidermis sheet was calculatedand expressed as a mean of density of three images.

Example 1 The A3 Adenosine Receptor Agonist IB-MECA Exerts a StimulatoryEffect on Melanin Synthesis and Secretion in B16 Melanocytes

Treatment of B16 melanocytes with increasing doses of IB-MECA resultedin a dose dependent increase of the melanin level, as shown in FIG. 1.Specifically, as shown in FIG. 1A, at the highest IB-MECA concentration,the intracellular melanin level was increased by 143±4.1% while thesecretion of melanin to the media was increased by 554±23.8% (p<0.001).As shown in FIG. 1B, exposure of the cells to 5 μM IB-MECA resulted inan increase of the total melanin level by 220±19.4% (p<0.001), whiletreatment with 10 μM of IB-MECA increased the total level by 369±1%, andtreatment with 40 μM of IB-MECA increased the melanin level by441±53.8%. Accordingly, the stimulatory effect of IB-MECA on the melaninlevel was attributable mainly to the increase in the secretion ofmelanin by B16 melanocytes (FIGS. 1A-1C).

Example 2 The A3 Adenosine Receptor Antagonist MRS-1523 Exerts anInhibitory Effect on Melanin Synthesis in B16 Melanocytes

MRS-1523 affected both the synthesis and the secretion of melanin by thecells as shown in FIG. 2. Exposure of melanocytes to 40 μM MRS-1523caused a decrease in total melanin by 67±1.07% (p<0.001; FIG. 2B). Thistreatment effectively blocked melanin secretion by 88±1.7%, while themelanin level within the cells decreased only by 11±2.3% (FIG. 2A).Similarly, upon treatment of the cells with 1 μM and 5 μM MRS-1523, themelanin level in the cells decreased only by 27±5.7% and 24±1.7%respectively. However, this treatment was associated with a significantdecrease of the intracellular melanin (48%±4.9, p<0.05). At allconcentrations tested within the range of 1-40 μM MRS-1523, the secretedand the total melanin levels were lower than that of the control(p<0.05, FIGS. 2A and 2B).

At lower concentrations of MRS-1523 the modulating effect on melaninsynthesis and secretion was less pronounced. Thus, the total melanin inmelanocytes exposed to 1 μM and 5 μM MRS-1523 decreased by 37±5.7% and24±1.9% respectively (p<0.05). At all MRS-1523 concentrations tested thesecreted and the total melanin (melanin in cell and in media) were lowerthan that of the control (p<0.05, FIGS. 2A and 2B).

Example 3 Time-Dependent Melanin Synthesis and Secretion by B16Melanocytes Treated with IB-MECA or MRS-1523

B16 melanocytes were exposed to 10 μM of IB-MECA or 10 μM MRS-1523 andthe melanin levels in the cells and in the media were monitored atdifferent time points following exposure to the ligands. The resultsindicated that detectable levels of melanin secreted to the medium(extracellular) were not observed after 24 hours of exposure to eitherIB-MECA or MRS-1523. However, as shown in. FIG. 3A, confocal microscopyshowed observable changes in melanin level and distribution in thecytosol of the treated cells. Specifically, more melansomes wereobserved in the cell periphery and in the dendritic sites of the controland IB-MECA treated melanocytes as compared to MRS-1523 treated cells(melansomes indicated by arrows). Moreover, in cells treated withIB-MECA, melanosomes were accumulated in the perinuclear areas(melansomes indicated by arrows). FIG. 3B depicts transfer ofmelanosomes from one melanocyte to the other by a dendrite-dendriteinteraction. Moreover the melanosomes appear to be injected and movedalong the edge of cells

FIG. 3C shows total melanin levels after various intervals of exposureto IB-MECA or MRS-1523. After 72 hours of exposure to IB-MECA, the levelof total melanin was non-significantly increased. However, after fourdays (96 h) of exposure to IB-MECA, the total melanin level (i.e.melanin in the cells and melanin secreted to the media) wassignificantly increased by 198±12.1% (p<0.05). Similarly, after fourdays (96 h) of exposure to MRS-1523, the total melanin level wassignificantly decreased by 67±4.4% (p<0.05). Exposure to IB-MECA for aperiod of 120 h resulted in an increase of 537±31.9% in the totalmelanin level; this increase was attributed mainly to the enhancedsecretion of melanin into the media (FIGS. 3C and 3D). A decrease of88±1.3% in the overall melanin level after 5 days of cells exposure toMRS-1523 was attributed to a similar decrease in melanin level in boththe cells and the media (p<0.001).

FIG. 3E is a photograph showing melanin secretion to the media incontrol cells and in cells exposed to IB-MECA or MRS-1523 for 5 days.Consistent with the results shown in FIGS. 3C and 3D, prolonged exposureof melanocytes to IB-MECA resulted in a visually observable increase inmelanin, whereas prolonged exposure of cells to MRS-1523 resulted in avisually observable decrease in melanin.

Example 4 The Effect of Kojic Acid on Melanin Levels, Synthesis andSecretion in B16 Melanocytes

Kojic acid is a well known melanization decreasing agent. The melaninsynthesis in cells has been well studied; however, no informationregarding the effect of kojic acid on melanin secretion, whichconstitutes a major contribution to skin melanization, was obtained. Inorder to evaluate the effect of kojic acid on the synthesis andsecretion of melanin in cells, B16 melanocytes were cultured in 10% DMEMin the presence of different concentrations of kojic acid for 5 days.The melanin and protein content were measured as described in Materialsand Methods.

As shown in FIG. 4B, kojic acid exerted inhibition of 51%±2.4 in thetotal melanin level (p<0.001). FIG. 4A shows that both melanin synthesis(overall melanin levels) and secretion (melanin level in the medium)were affected.

Example 5 The Effect of A3 Adenosine Receptor Ligands on AktPhosphorylation

In order to explore the possible mechanisms involved in the modulationof melanin synthesis, exerted by IB-MECA in increasing melanin levels,and by MRS-1523 in decreasing melanin levels, the effects of short timeexposure to both agents on Akt phosphorylation in B16 melanocytes weretested. Serum starved B16 melanocytes were exposed to 10 μM IB-MECA orMRS-1523 10-180 minutes, as indicated in FIGS. 5A and 5B. Cells werecollected and protein was extracted and quantified as described inMaterials and Methods. Proteins were analyzed for Akt phosphorylationand total Akt expression by western blot analysis (FIG. 5A). Cellstreated for 3 h were analyzed by confocal microscopy (FIG. 5B).

The results shown in FIG. 5A indicate that both the A3 adenosinereceptor agonist (IB-MECA) and the A3 adenosine receptor antagonist(MRS-1523) induced phosphorylation of Akt within 10 min. However,continued exposure to MRS-1523 (up to 3 h) led to downregulation ofP-Akt (FIG. 5A left panel), while continued exposure to IB-MECA resultedin increased phosphorylation of Akt (FIG. 5A right panel).

The Akt family signal pathway has been shown to be upstream to the ERKsignal pathway, and the increase in phosphorylated Akt is implicated indownregulation of phosphorylated ERK. Without wishing to be bound by anyparticular mechanism and theory, it is contemplated that increasing thelevel of phosphorylated ERK leads to downregulation of the transcriptionfactor Mitf which regulates tyrosinase, a key enzyme in melanogenesisand cell differentiation.

Example 6 The Effect of A3 Adenosine Receptor Ligands on ERKPhosphorylation

The ERK signaling pathway has been shown to be upstream to thetranscription factor Mitf and downstream to the Akt signaling pathway.ERK phosphorylation analyses show that MRS-1523 induced phophorylationof ERK 1 within 30 minutes and phosphorylation of ERK 2 within 10minutes (FIG. 6A). In contrast, IB-MECA (10 μM) did not increase thelevel of ERK phosphorylation (FIG. 6A).

ERK phosphorylation in non-starved cultures (containing 10% FCS) after72 hours of exposure to the ligands was examined. Exposure to MRS-1523,stimulated phosphorylation of both ERK1 and ERK2, while exposure toIB-MECA did not appear to affect ERK phosphorylation. (FIG. 6B).

Example 7 The Effect of A3 Adenosine Ligands on Mitf Expression andLocalization in B16 Melanocytes

Mitf is a transcription factor which regulates tyrosinase, a key enzymein melanogenesis and cell differentiation and has been shown to bedownstream to ERK. FIG. 7 shows that Mitf is highly expressed in thecontrol and IB-MECA treated cells (FIG. 7, left and middle panels).Moreover, exposure of cells to the A3 adenosine receptor agonist IB-MECAinduced translocation of Mitf protein to the nucleus within 3 h ofexposure, while in control cells Mitf remained in the cytosol. After 24h of exposure to A3 adenosine receptor agonist, the expression of Mitfin IB-MECA treated cells remained high, both in the cytoplasm and in thenucleus. In contrast, in the control cells Mitf expression was observedmainly in the cytosol. Furthermore, cells treated with the A3 adenosinereceptor antagonist MRS-1523 exhibited very low Mitf expression both inthe cytosol and in the nucleus (FIG. 7, right panels).

Without wishing to be bound by any particular mechanism or theory, theresults disclosed herein support the conclusion that the effects ofenhanced pigmentation exerted by IB-MECA and decreased pigmentationexerted by MRS-1523, are respectively mediated by Mitf activation andMitf downregulation, via the Akt-ERK-Mitf signaling pathway.

Example 8 The Effect of A3 Adenosine Receptor Ligands on Ex Vivo HumanSkin Pigmentation

The results shown in FIGS. 8A-8E indicate that topical treatment ofhuman skin explants with the A3 adenosine receptor antagonist MRS-1523decreased melanin levels, whereas treatment with the A3 adenosinereceptor agonist IB-MECA increased melanin levels, both after 12 days ofexposure. It was further observed that hydroquinone, usually recognizedas a whitening agent, increased skin pigmentation when applied topicallyat a concentration of 100 μM for 12 days.

FIGS. 8B and 8D show the increased number of dark-stained DOPA positivemelanocytes (D-P-M) in. IB-MECA-treated and hydroquinone-treated samplesas compared to control. FIGS. 8B and 8D further show the decreasednumber of D-P-M in MRS-1523-treated samples as compared to control.

Evaluation of pigmentation level following DOPA staining, as measured bylight transmission though the epidermal sheets, indicated that MRS-1523decreased pigmentation by 80+21.8% and 49.5+6.9% respectively at ligandconcentrations of 10 μM and 50 μM, whereas IB-MECA increasedpigmentation by 27+3.9% and 36+2.6% respectively at concentrations of 10μM and 50 μM (FIGS. 8C and 8E).

Example 9 Effect of A3 Adenosine Receptor Ligands on Skin PigmentationEvaluated by Fontana-Masson Stain

Histological analysis of paraffin embedded and Fontana-Masson stainedskin sections showed increased pigmentation in skin samples treated withthe A3 adenosine receptor agonist IB-MECA (10 μM) or with the knownmelanogenesis enhancing agent 100 μM IBMX (100 μM), compared to control,and decreased pigmentation in skin samples treated with the A3 adenosinereceptor antagonist. MRS-1523 (FIG. 9). IBMX(3-isobutyl-1-methyl-xanthine) was used at a concentration of 100 μM asa positive control for achieving an enhanced pigmentation effect (seeGibbs et al., Pigment Cell Res. December 2000; 13(6):458-66).

The histological appearance of IB-MECA-treated samples suggests that theobserved increase in skin pigmentation is due to an elevation in melaninaccumulation and/or deposition in the adjacent keratinocytes, ratherthan an increase in the number of melanocytes (see FIG. 9A). Incontrast, the melanocytes in MRS-1523-treated cells appear to containless melanin and there is a lesser extent of melanin accumulation and/ordeposition in the keratinocytes (see FIG. 9D).

The results disclosed herein, which are based on observations in humanskin, are consistent with the results of the in vitro studies describedin Examples 1-3, showing elevation of melanin secretion into theextracellular medium following treatment with IB-MEGA. That is,increased secretion may account for increased amounts of melanindeposited in the adjacent keratinocytes in the ex vivo human skin organculture.

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While certain embodiments of the invention have been illustrated anddescribed, it will be clear that the invention is not limited to theembodiments described herein. Numerous modifications, changes,variations, substitutions and equivalents will be apparent to thoseskilled in the art without departing from the spirit and scope of thepresent invention as described by the claims, which follow.

1-52. (canceled)
 53. A method for modulating melanin production,secretion, accumulation, or a combination thereof, comprising contactinga skin cell with an effective amount of an A3 adenosine receptorantagonist, or an A3 adenosine receptor agonist.
 54. The methodaccording to claim 53, wherein said skin cell is a melanocyte or akeratinocyte.
 55. The method according to claim 53, wherein said skincell is within a tissue which comprises a plurality of melanocytes,keratinocytes, or both.
 56. The method according to claim 53, whereinsaid A3 adenosine receptor antagonist decreases melanin production,secretion, accumulation, or combination thereof in said skin cell. 57.The method according to claim 53, wherein said A3 adenosine receptorantagonist decreases secretion of melanin from a melanocyte anddeposition of said melanin in a keratinocyte.
 58. The method accordingto claim 53, wherein said A3 adenosine receptor antagonist is a compoundselected from the group consisting of: a dihydropyridine, a pyridine, apyridinium salt, a triazoloquinazoline, an imidazoquinoline andderivatives, or analogs thereof.
 59. The method according to claim 53,wherein said A3 adenosine receptor antagonist is selected from the groupconsisting of:3-propyl-6-ethyl-5[(ethylthio)carbonyl]-2-phenyl-4-propyl-3-pyridinecarboxylate (MRS-1523);1,4-dihydro-2-methyl-6-phenyl-4-(phenylethynyl)-3,5-pyridinedicarboxylicacid 3-ethyl-5-[(3-nitrophenyl)methyl]ester (MRS-1334);3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(±)-dihydropyridine-3,5dicarboxylate (MRS-1191); and3-ethyl-5-benzyl-2-methyl-6-phenyl-4-styryl-1,4-(±)-dihydropyridine-3,5-dicarboxylate(MRS-1097), and3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(±)-dihydropyridine-3,5dicarboxylate (MRS-1191).
 60. The method according to claim 56, furthercomprises contacting said skin cell with: hydroxytetronic acid, tetronicacid, hydroquinone, an α-hydroxy acid, a fatty acid ester of ascorbicacid, a tyrosinase inhibitor, a tyrosine phosphatase inhibitor, or anycombination thereof.
 61. The method according to claim 53, wherein saidA3 adenosine receptor antagonist is suitable for application to a skinof a subject afflicted with a hyper-pigmentation disorder selected fromthe group consisting of: pigmented spots, lentigo senilis, freckles,café au lait spots, liver spots, ephelides, periorbital darkening,post-inflammatory hyper-pigmentation, pigmented keratosis, melasma,chloasma, and hyper-pigmentation due to a skin graft procedure.
 62. Themethod according to claim 53, wherein said A3 adenosine receptor agonistincreases melanin production, secretion, accumulation, or anycombination thereof in said skin cell.
 63. The method according to claim53, wherein said A3 adenosine receptor agonist increases secretion ofmelanin from a melanocyte and deposition of said melanin in akeratinocyte.
 64. The method according to claim 53, wherein said A3adenosine receptor agonist is an N6-substituted-adenosine-5′-uronamide.65. The method according to claim 64, wherein saidN6-substituted-adenosine-5′-uronamide is selected from the groupconsisting of: an N6-benzyladenosine-5′-uronamide, anN6-4-substituted-sulfonamidophenylcarbamoyl-adenosine-5′-uronamide, a2-chloro-N6-substituted-4′-thioadenosine-5′-uronamide; an(N)-methanocarba adenosine-5′-uronamide, and derivatives or analogsthereof.
 66. The method according to claim 53, wherein said A3 adenosinereceptor agonist is selected from the group consisting of:N6-(3-iodobenzyl) adenosine-5′-(N-methyluronamide) (IB-MECA),2-chloro-N6-(3-iodobenzyl)-adenosine-5′-(N-methyluronamide)(Cl-IB-MECA), N6-(4-amino-3-iodobenzyl)-adenosine-5′-(N-methyluronamide)(AB-MECA), and N6-2-(4-aminophenyl) ethyladenosine (APNEA).
 67. Themethod according to claim 62, further comprises contacting said skincell with an allosteric enhancer of the A3 adenosine receptor.
 68. Themethod according to claim 53, wherein said A3 adenosine receptor agonistis suitable for application to a skin of a subject afflicted with ahypo-pigmentation disorder selected from the group consisting of:vitiligo, piebaldism, leukoderma due to cicatrisation, nevusdepigmentosis, and depigmentation due to a skin graft procedure.